PROJECT SUMMARY/ABSTRACT Type 1 Diabetes Mellitus (T1D) affects millions of individuals world-wide. Disappointing results of clinical trials using immunomodulatory drug treatments have identified a critical need for alternative treatment approaches and earlier identification of T1D, at a time prior to irremediable dysfunction or destruction of pancreatic ? cells. Recent data indicate that intrinsic ? cell responses to inflammatory and metabolic stress ultimately impact ? survival, and emerging work suggests that ? cell extracellular vesicles (EVs), membrane bound nanoparticles including exosomes, microvesicles, and apoptotic bodies, play an important role in T1D pathogenesis. Preliminary data generated as part of the applicant's K08 award demonstrate that ? cell and circulating EV cargo are altered in T1D. Based on this, our central hypothesis is that ? cell EVs play a pathophysiologic role in the development of T1D, with altered EV cargo before and after T1D development. The long-term goals of this applicant are to define the role of ? cell EVs in diabetes pathophysiology and to use changes in circulating ? cell EV cargo as the basis for biomarkers that successfully identify the onset of T1D in prediabetic individuals. In Specific Aim 1, we will address the underlying mechanism of alterations in EV microRNA cargo during T1D development by testing the working hypothesis that cytokine-induced increases in ? cell exosome miRNAs are influenced by the ceramide generating enzyme, neutral sphingomyelinase 2. This hypothesis will be tested using chemical and genetic inhibition and overexpression of this enzyme in clonal human ? cells and islets. In Specific Aim 2, we propose experiments to develop and test ? cell-specific EV targets using both directed and unbiased approaches based on the working hypothesis that proteins specific to ? cells will also be present in ? cell EVs, and could therefore allow for selective isolation of ? cell EVs. In addition to careful ex-vivo study of candidate reagents, we will test feasibility of this approach using an in-vivo xenotransplant model. Completion of these experiments will address an important mechanistic question regarding ? cell EV biology and identify tools that may be harnessed to isolate circulating ? cell or islet-derived EVs. The molecular biology expertise accumulated through experiments and training in the applicant's K08 award and the research environment of the Indiana University Center for Diabetes and Metabolic Diseases make the applicant uniquely suited to execute the Aims of this proposal. Importantly, this proposal has also been designed to generate data and resources that the applicant will utilize in an R01 application defining the physiologic significance and clinical biomarker potential of ? cell EV miRNAs during T1D development.